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  • 96
    Thermo Fisher trizol reagent
    RT-PCR analysis of defensin mRNA expression by primary epithelial cells . Primary epithelial cells were obtained from human nasal turbinates (HNT), as described in Methods. The cells (5 × 10 6 ) were grown in the six well plates for 48 hours. The cells were then exposed to either the latex beads or A. fumigatus organisms for 18 hours. The mRNA was then isolated by <t>TRIzol</t> Reagent and RT-PCR was performed as described above in Materials and Methods. Specific primer pairs (Table 1) were used for <t>RNA</t> amplification. The sizes of amplified products are indicated and were as predicted. The hBD2 and hBD9 products were sequenced and confirmed to be identical to the predicted sequence. GAPDH was uniformly expressed. Cells in a control well were cultivated in the absence of A. fumigatus . One of the three results is shown.
    Trizol Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 96 stars, based on 14 article reviews
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    trizol reagent - by Bioz Stars, 2019-10
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    77
    Roche trizol reagent
    CO-EtOAc regulates the expression of lipid metabolism-related genes in FL83B hepatocytes. Total RNAs were extracted using <t>Trizol</t> reagent and mRNA was measured using <t>qRT-PCR.</t> Relative mRNA levels were normalized to the housekeeping gene, r18S. The fold changes relative to the control were calculated using the ΔΔCT method for mRNA expression levels of fatty acid transporter genes, CD36 and FABP ; lipogenic genes: SREBP-1c , FAS , and ACC1 ; fatty acid oxidation genes: PPAR-α, CPT-1 , and ACOX . All experimental groups had been treated with the same concentration of DMSO. ( A ) Cells were incubated with different concentrations (50–400 μg/mL) of CO-EtOAc. ( B ) Cells were incubated with 600 μM OA in the presence or absence of different concentrations (50–400 μg/mL) of CO-EtOAc. All results are presented as the mean ± SEM of three independent experiments. Values are expressed as relative fold change compared with the control group, which is set as 1. * p
    Trizol Reagent, supplied by Roche, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/trizol reagent/product/Roche
    Average 77 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    trizol reagent - by Bioz Stars, 2019-10
    77/100 stars
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    RT-PCR analysis of defensin mRNA expression by primary epithelial cells . Primary epithelial cells were obtained from human nasal turbinates (HNT), as described in Methods. The cells (5 × 10 6 ) were grown in the six well plates for 48 hours. The cells were then exposed to either the latex beads or A. fumigatus organisms for 18 hours. The mRNA was then isolated by TRIzol Reagent and RT-PCR was performed as described above in Materials and Methods. Specific primer pairs (Table 1) were used for RNA amplification. The sizes of amplified products are indicated and were as predicted. The hBD2 and hBD9 products were sequenced and confirmed to be identical to the predicted sequence. GAPDH was uniformly expressed. Cells in a control well were cultivated in the absence of A. fumigatus . One of the three results is shown.

    Journal: BMC Microbiology

    Article Title: Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms

    doi: 10.1186/1471-2180-9-33

    Figure Lengend Snippet: RT-PCR analysis of defensin mRNA expression by primary epithelial cells . Primary epithelial cells were obtained from human nasal turbinates (HNT), as described in Methods. The cells (5 × 10 6 ) were grown in the six well plates for 48 hours. The cells were then exposed to either the latex beads or A. fumigatus organisms for 18 hours. The mRNA was then isolated by TRIzol Reagent and RT-PCR was performed as described above in Materials and Methods. Specific primer pairs (Table 1) were used for RNA amplification. The sizes of amplified products are indicated and were as predicted. The hBD2 and hBD9 products were sequenced and confirmed to be identical to the predicted sequence. GAPDH was uniformly expressed. Cells in a control well were cultivated in the absence of A. fumigatus . One of the three results is shown.

    Article Snippet: Total RNA was isolated with TRIzol Reagent (Invitrogen, Cat N 15596-026) according to the manufacturer's instructions.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Amplification, Sequencing

    RT-PCR analysis of defensin expression by 16HBE cells exposed to A. fumigatus organisms in the presence of different serums . 16HBE human epithelial bronchial cells (5 × 10 6 ) were grown in six well plates for 24 hours. The cells were then exposed to the different morphotypes of A. fumigatus or the latex beads in the presence of either Human (HS) or Fetal Calf Serum (FCS), (heated or not at 56°C). After 18 hours of incubation, the cells were washed with PBS, mRNA was isolated by TRIzol Reagent, and RT-PCR was performed as described above in Materials and Methods. Specific primer pairs (Table 1) were used for RNA amplification. The sizes of amplified products are indicated and were as predicted. All products were amplified according to the conditions described in Table 1. Cells were cultivated in a control well in the absence of A. fumigatus . GAPDH was uniformly expressed. One of the four results is shown.

    Journal: BMC Microbiology

    Article Title: Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms

    doi: 10.1186/1471-2180-9-33

    Figure Lengend Snippet: RT-PCR analysis of defensin expression by 16HBE cells exposed to A. fumigatus organisms in the presence of different serums . 16HBE human epithelial bronchial cells (5 × 10 6 ) were grown in six well plates for 24 hours. The cells were then exposed to the different morphotypes of A. fumigatus or the latex beads in the presence of either Human (HS) or Fetal Calf Serum (FCS), (heated or not at 56°C). After 18 hours of incubation, the cells were washed with PBS, mRNA was isolated by TRIzol Reagent, and RT-PCR was performed as described above in Materials and Methods. Specific primer pairs (Table 1) were used for RNA amplification. The sizes of amplified products are indicated and were as predicted. All products were amplified according to the conditions described in Table 1. Cells were cultivated in a control well in the absence of A. fumigatus . GAPDH was uniformly expressed. One of the four results is shown.

    Article Snippet: Total RNA was isolated with TRIzol Reagent (Invitrogen, Cat N 15596-026) according to the manufacturer's instructions.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation, Isolation, Amplification

    RT-PCR analysis of various defensin expression levels in human 16HBE epithelial bronchial cells exposed to A. fumigatus organisms . 16HBE human epithelial tracheal cells (5 × 10 6 ) were grown in six well plates for 24 hours. After exposing the cells to RC, SC, HF or latex beads for 18 hours, the cells were washed with PBS, mRNA was isolated by TRIzol Reagent and RT-PCR was performed as described above in Materials and Methods. Specific primer pairs (Table 1) were used for RNA amplification: hBD1, 273 bp product; hBD2, 199 bp product; hBD8, 176 bp product; hBD9, 174 bp product; hBD18, 400 bp product and human GAPDH, which was used as an internal control, 473-bp product. All products were amplified according to the conditions described in Table 1. Cells were cultivated in a control well in the absence of A. fumigatus . As a positive control for defensin expression, exposure to human Il-1β was used in all experiments. The hBD1, hBD2 and hBD9 products were sequenced and confirmed to be identical to the predicted sequence. GAPDH was uniformly expressed. One of the four experiments is shown. Abbreviations: resting conidia (RC), swollen conidia (SC), hyphal fragments (HF), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), interleukin-1β (Il–1β).

    Journal: BMC Microbiology

    Article Title: Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms

    doi: 10.1186/1471-2180-9-33

    Figure Lengend Snippet: RT-PCR analysis of various defensin expression levels in human 16HBE epithelial bronchial cells exposed to A. fumigatus organisms . 16HBE human epithelial tracheal cells (5 × 10 6 ) were grown in six well plates for 24 hours. After exposing the cells to RC, SC, HF or latex beads for 18 hours, the cells were washed with PBS, mRNA was isolated by TRIzol Reagent and RT-PCR was performed as described above in Materials and Methods. Specific primer pairs (Table 1) were used for RNA amplification: hBD1, 273 bp product; hBD2, 199 bp product; hBD8, 176 bp product; hBD9, 174 bp product; hBD18, 400 bp product and human GAPDH, which was used as an internal control, 473-bp product. All products were amplified according to the conditions described in Table 1. Cells were cultivated in a control well in the absence of A. fumigatus . As a positive control for defensin expression, exposure to human Il-1β was used in all experiments. The hBD1, hBD2 and hBD9 products were sequenced and confirmed to be identical to the predicted sequence. GAPDH was uniformly expressed. One of the four experiments is shown. Abbreviations: resting conidia (RC), swollen conidia (SC), hyphal fragments (HF), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), interleukin-1β (Il–1β).

    Article Snippet: Total RNA was isolated with TRIzol Reagent (Invitrogen, Cat N 15596-026) according to the manufacturer's instructions.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Amplification, Positive Control, Sequencing

    Analysis of the defensin expression and its localisation in pneumocytes A549 exposed to live A. fumigatus . A . RT-PCR analysis of defensin mRNA expression by human pneumocyte A549 cells exposed to live A. fumigatus . A549 human epithelial bronchial cells (5 × 10 6 ) were grown in six well plates for 24 hours. The cells were then exposed either to live A. fumigatus conidia or latex beads. After 18 hours of incubation, the cells were washed with PBS, mRNA was isolated by TRIzol Reagent, and RT-PCR was performed as described above in Methods. Specific primer pairs (Table 1) were used for RNA amplification. The size of the amplified product is indicated and was as predicted. Cells were cultivated in a control well in the absence of A. fumigatus . As an additional control, the cells were exposed to 10 6 latex beads for the same period. GAPDH was uniformly expressed. One of the four results is shown. B . Immunofluorescence detection of hBD2 in the A549 exposed to live A. fumigatus conidia. A549 cells were seeded at 5 × 10 5 cells per well in 1 ml of DMEM/F12 on 18-mm-diameter cover slips in 12 well plates in triplicate and grown for 16 h at 37°C. After washing the cover slips with 1%BSA/PBS, the cells were exposed to either latex beads or live A. fumigatus conidia for 18 hours. Il-1β was used as a positive control. Some cells were treated with TNF-α. Following washing with PBS, the cells were fixed with a paraformaldehyde solution for 30 min at 37°C. The slides were then incubated in 1% BSA/PBS, followed by a solution of 10% normal goat serum. After washing, polyclonal rabbit anti-human hBD2 at a dilution of 1:250 was applied as primary antibody overnight at 4°C, followed by incubation with FITC-labelled goat anti-rabbit secondary antibody at a dilution of 1:300 for 4 hours at room temperature. After washing, the cover slips were mounted on slides with ProLong antifade Vectashield. Samples were viewed with a Zeiss fluorescence microscope using ×400 magnification. The arrows indicate the cells stained with anti-hBD2 antibody. The percentage of stained cells was computed from triplicates of four experiments. Means followed by the same letter are not significantly different. +, presence; -, absence of Il-1β, TNF-α, live A. fumigatus organism and latex beads. The punctuated localisation of the signal, which is concentrated adjacent to the nucleus (arrow), was observed. The data shown are representative of four independent experiments.

    Journal: BMC Microbiology

    Article Title: Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms

    doi: 10.1186/1471-2180-9-33

    Figure Lengend Snippet: Analysis of the defensin expression and its localisation in pneumocytes A549 exposed to live A. fumigatus . A . RT-PCR analysis of defensin mRNA expression by human pneumocyte A549 cells exposed to live A. fumigatus . A549 human epithelial bronchial cells (5 × 10 6 ) were grown in six well plates for 24 hours. The cells were then exposed either to live A. fumigatus conidia or latex beads. After 18 hours of incubation, the cells were washed with PBS, mRNA was isolated by TRIzol Reagent, and RT-PCR was performed as described above in Methods. Specific primer pairs (Table 1) were used for RNA amplification. The size of the amplified product is indicated and was as predicted. Cells were cultivated in a control well in the absence of A. fumigatus . As an additional control, the cells were exposed to 10 6 latex beads for the same period. GAPDH was uniformly expressed. One of the four results is shown. B . Immunofluorescence detection of hBD2 in the A549 exposed to live A. fumigatus conidia. A549 cells were seeded at 5 × 10 5 cells per well in 1 ml of DMEM/F12 on 18-mm-diameter cover slips in 12 well plates in triplicate and grown for 16 h at 37°C. After washing the cover slips with 1%BSA/PBS, the cells were exposed to either latex beads or live A. fumigatus conidia for 18 hours. Il-1β was used as a positive control. Some cells were treated with TNF-α. Following washing with PBS, the cells were fixed with a paraformaldehyde solution for 30 min at 37°C. The slides were then incubated in 1% BSA/PBS, followed by a solution of 10% normal goat serum. After washing, polyclonal rabbit anti-human hBD2 at a dilution of 1:250 was applied as primary antibody overnight at 4°C, followed by incubation with FITC-labelled goat anti-rabbit secondary antibody at a dilution of 1:300 for 4 hours at room temperature. After washing, the cover slips were mounted on slides with ProLong antifade Vectashield. Samples were viewed with a Zeiss fluorescence microscope using ×400 magnification. The arrows indicate the cells stained with anti-hBD2 antibody. The percentage of stained cells was computed from triplicates of four experiments. Means followed by the same letter are not significantly different. +, presence; -, absence of Il-1β, TNF-α, live A. fumigatus organism and latex beads. The punctuated localisation of the signal, which is concentrated adjacent to the nucleus (arrow), was observed. The data shown are representative of four independent experiments.

    Article Snippet: Total RNA was isolated with TRIzol Reagent (Invitrogen, Cat N 15596-026) according to the manufacturer's instructions.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Isolation, Amplification, Immunofluorescence, Positive Control, Fluorescence, Microscopy, Staining

    Inhibition of HCV1b attachment to DHHs by purified HSPG (A) and Heparin (B). The HCV1b was pre-incubated with varying amounts of HSPG or Heparin for 1 hr on ice prior to adding to day-11 DHHs in 12-well cell culture plates as described in materials and methods . After incubation on ice for 2 hrs, the unbound HCV was removed by washing cells with PBS for three times. The vRNA of the cell-bound HCV was extracted with Trizol reagent (Invitrogen). The levels of HCV1b vRNA were determined using the same real-time RT-qPCR method as in Fig. 1.

    Journal: PLoS ONE

    Article Title: Apolipoprotein E Mediates Attachment of Clinical Hepatitis C Virus to Hepatocytes by Binding to Cell Surface Heparan Sulfate Proteoglycan Receptors

    doi: 10.1371/journal.pone.0067982

    Figure Lengend Snippet: Inhibition of HCV1b attachment to DHHs by purified HSPG (A) and Heparin (B). The HCV1b was pre-incubated with varying amounts of HSPG or Heparin for 1 hr on ice prior to adding to day-11 DHHs in 12-well cell culture plates as described in materials and methods . After incubation on ice for 2 hrs, the unbound HCV was removed by washing cells with PBS for three times. The vRNA of the cell-bound HCV was extracted with Trizol reagent (Invitrogen). The levels of HCV1b vRNA were determined using the same real-time RT-qPCR method as in Fig. 1.

    Article Snippet: The vRNA of the cell-bound HCV was extracted with Trizol Reagent (Invitrogen) and quantified by qRT-PCR using the StepOnePlus real-time PCR system.

    Techniques: Inhibition, Purification, Incubation, Cell Culture, Quantitative RT-PCR

    Blockade of HCV1b cell attachment by apoE monoclonal antibody mAb23. DHHs at day-11 were incubated with HCV1b in the absence (Control) or presence of 10 µg/ml of normal mouse IgG1 (mIgG1) or increasing amounts of apoE mAb23 (0.4, 2, and 10 µg/ml) at 37°C for 2 hrs. The unbound HCV was removed by washing cells with 1x PBS for three times. The vRNA of the cell-bound HCV was extracted with Trizol reagent (Invitrogen). The levels of HCV vRNA were quantified by a real-time RT-PCR method using SuperScript® III Platinum® SYBR® Green One-Step qPCR Kit (Invitrogen). Reactions were run in a StepOnePlus real-time PCR system (Applied Biosystems) using the conditions provided by the qPCR kit. A house-keeping gene GAPDH was used as an internal control, which was quantified using Hu-GAPDH primer/probe mix containing vic-TAMRA (Applied Biosystems). The levels of HCV vRNA were calculated from the average data of three experiments upon normalization with the level of GAPDH.

    Journal: PLoS ONE

    Article Title: Apolipoprotein E Mediates Attachment of Clinical Hepatitis C Virus to Hepatocytes by Binding to Cell Surface Heparan Sulfate Proteoglycan Receptors

    doi: 10.1371/journal.pone.0067982

    Figure Lengend Snippet: Blockade of HCV1b cell attachment by apoE monoclonal antibody mAb23. DHHs at day-11 were incubated with HCV1b in the absence (Control) or presence of 10 µg/ml of normal mouse IgG1 (mIgG1) or increasing amounts of apoE mAb23 (0.4, 2, and 10 µg/ml) at 37°C for 2 hrs. The unbound HCV was removed by washing cells with 1x PBS for three times. The vRNA of the cell-bound HCV was extracted with Trizol reagent (Invitrogen). The levels of HCV vRNA were quantified by a real-time RT-PCR method using SuperScript® III Platinum® SYBR® Green One-Step qPCR Kit (Invitrogen). Reactions were run in a StepOnePlus real-time PCR system (Applied Biosystems) using the conditions provided by the qPCR kit. A house-keeping gene GAPDH was used as an internal control, which was quantified using Hu-GAPDH primer/probe mix containing vic-TAMRA (Applied Biosystems). The levels of HCV vRNA were calculated from the average data of three experiments upon normalization with the level of GAPDH.

    Article Snippet: The vRNA of the cell-bound HCV was extracted with Trizol Reagent (Invitrogen) and quantified by qRT-PCR using the StepOnePlus real-time PCR system.

    Techniques: Cell Attachment Assay, Incubation, Quantitative RT-PCR, SYBR Green Assay, Real-time Polymerase Chain Reaction

    Effect of heparinase treatment on HCV1b attachment to DHHs. The day-11 DHHs in 12-well cell culture plates were incubated with varying concentrations of heparinase I in a buffer containing 20 mM Tris-HCl (pH 6.8), 50 mM NaCl, 4 mM CaCl 2 and 0.01% bovine serum albumin at 37°C for 1 hr [18] . The heparinase-treated DHHs were then incubated with HCV1b on ice for 2 hrs. The unbound HCV was removed and the cells were washed with 1x PBS for three times. The HCV1b vRNA of the cell-bound HCV was extracted with Trizol reagent and quantified by RT-qPCR using the StepOnePlus real-time PCR system same as that in Fig. 1.

    Journal: PLoS ONE

    Article Title: Apolipoprotein E Mediates Attachment of Clinical Hepatitis C Virus to Hepatocytes by Binding to Cell Surface Heparan Sulfate Proteoglycan Receptors

    doi: 10.1371/journal.pone.0067982

    Figure Lengend Snippet: Effect of heparinase treatment on HCV1b attachment to DHHs. The day-11 DHHs in 12-well cell culture plates were incubated with varying concentrations of heparinase I in a buffer containing 20 mM Tris-HCl (pH 6.8), 50 mM NaCl, 4 mM CaCl 2 and 0.01% bovine serum albumin at 37°C for 1 hr [18] . The heparinase-treated DHHs were then incubated with HCV1b on ice for 2 hrs. The unbound HCV was removed and the cells were washed with 1x PBS for three times. The HCV1b vRNA of the cell-bound HCV was extracted with Trizol reagent and quantified by RT-qPCR using the StepOnePlus real-time PCR system same as that in Fig. 1.

    Article Snippet: The vRNA of the cell-bound HCV was extracted with Trizol Reagent (Invitrogen) and quantified by qRT-PCR using the StepOnePlus real-time PCR system.

    Techniques: Cell Culture, Incubation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    Inhibition of HCV1b attachment to DHHs by apoE-derived or HSPG-binding peptide. The day-11 DHHs in 12-well cell culture plates were incubated with HCV1b in the absence or presence of increasing concentrations (0, 6.7, 20, and 60 µM) of peptides on ice for 2 hrs. Upon removal of unbound HCV1b by extensive washing with PBS, the vRNA of the cell-bound HCV1b was extracted with Trizol reagent. The levels of HCV1b vRNA were quantified by a real-time RT-qPCR method. A . Sequences of synthetic peptides. B . Inhibition of HCV1b cell attachment by a peptide derived from the apoE receptor-binding domain. C . Blockade of HCV1b cell attachment by an HSPG-binding peptide 6a-P. The relative levels of HCV1b vRNA are on average of three experiments were converted to percentage of control (%) considering the level of HCV vRNA in the absence of peptide 100%. The relative levels of HCV1b vRNA (y-axis) are plotted against concentrations of peptides (x-axis).

    Journal: PLoS ONE

    Article Title: Apolipoprotein E Mediates Attachment of Clinical Hepatitis C Virus to Hepatocytes by Binding to Cell Surface Heparan Sulfate Proteoglycan Receptors

    doi: 10.1371/journal.pone.0067982

    Figure Lengend Snippet: Inhibition of HCV1b attachment to DHHs by apoE-derived or HSPG-binding peptide. The day-11 DHHs in 12-well cell culture plates were incubated with HCV1b in the absence or presence of increasing concentrations (0, 6.7, 20, and 60 µM) of peptides on ice for 2 hrs. Upon removal of unbound HCV1b by extensive washing with PBS, the vRNA of the cell-bound HCV1b was extracted with Trizol reagent. The levels of HCV1b vRNA were quantified by a real-time RT-qPCR method. A . Sequences of synthetic peptides. B . Inhibition of HCV1b cell attachment by a peptide derived from the apoE receptor-binding domain. C . Blockade of HCV1b cell attachment by an HSPG-binding peptide 6a-P. The relative levels of HCV1b vRNA are on average of three experiments were converted to percentage of control (%) considering the level of HCV vRNA in the absence of peptide 100%. The relative levels of HCV1b vRNA (y-axis) are plotted against concentrations of peptides (x-axis).

    Article Snippet: The vRNA of the cell-bound HCV was extracted with Trizol Reagent (Invitrogen) and quantified by qRT-PCR using the StepOnePlus real-time PCR system.

    Techniques: Inhibition, Derivative Assay, Binding Assay, Cell Culture, Incubation, Quantitative RT-PCR, Cell Attachment Assay

    Effect of camel milk on apoptotic and oxidative stress markers Caspase-3 (a), DR4 (b), and HO-1 (c) mRNA levels in MCF7 cells. MCF7 cells were treated for 6 h with a various concentrations of camel milk (2.5, 5, 10, and 20 mg/mL). Thereafter, total RNA was isolated using TRIzol reagent and the mRNA levels of Caspase-3, DR4, and HO-1 were quantified using RT-PCR normalized to β -actin housekeeping gene as described Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Camel Milk Triggers Apoptotic Signaling Pathways in Human Hepatoma HepG2 and Breast Cancer MCF7 Cell Lines through Transcriptional Mechanism

    doi: 10.1155/2012/593195

    Figure Lengend Snippet: Effect of camel milk on apoptotic and oxidative stress markers Caspase-3 (a), DR4 (b), and HO-1 (c) mRNA levels in MCF7 cells. MCF7 cells were treated for 6 h with a various concentrations of camel milk (2.5, 5, 10, and 20 mg/mL). Thereafter, total RNA was isolated using TRIzol reagent and the mRNA levels of Caspase-3, DR4, and HO-1 were quantified using RT-PCR normalized to β -actin housekeeping gene as described Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P

    Article Snippet: Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions and quantified by measuring the absorbance at 260 nm.

    Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction

    Effect of camel milk on apoptotic markers Caspase-3 (a), p53 (b), BcL2 (c), and DR4 (d) mRNA levels in HepG2 cells. HepG2 cells were treated for 6 h with a various concentrations of camel milk (2.5, 5, 10, and 20 mg/mL). Thereafter, total RNA was isolated using TRIzol reagent, and the mRNA levels of Caspase-3, p53, BcL2, and DR4 were quantified using RT-PCR normalized to β -actin housekeeping gene as described in Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Camel Milk Triggers Apoptotic Signaling Pathways in Human Hepatoma HepG2 and Breast Cancer MCF7 Cell Lines through Transcriptional Mechanism

    doi: 10.1155/2012/593195

    Figure Lengend Snippet: Effect of camel milk on apoptotic markers Caspase-3 (a), p53 (b), BcL2 (c), and DR4 (d) mRNA levels in HepG2 cells. HepG2 cells were treated for 6 h with a various concentrations of camel milk (2.5, 5, 10, and 20 mg/mL). Thereafter, total RNA was isolated using TRIzol reagent, and the mRNA levels of Caspase-3, p53, BcL2, and DR4 were quantified using RT-PCR normalized to β -actin housekeeping gene as described in Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P

    Article Snippet: Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions and quantified by measuring the absorbance at 260 nm.

    Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction

    Effect of MAPKs inhibitors on camel milk mediated induction of caspase-3 mRNA levels in HepG2 cells. HepG2 cells were pre-treated with for 2 h with JNK inhibitor, SP600125, p38 inhibitor, SB203580, and ERK inhibitor, U0126, before the addition of camel milk (10 mg/mL) for additional 6 h. Thereafter, total RNA was isolated using TRIzol reagent and the mRNA levels of Caspase-3 were quantified using RT-PCR normalized to β -actin housekeeping gene as described Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Camel Milk Triggers Apoptotic Signaling Pathways in Human Hepatoma HepG2 and Breast Cancer MCF7 Cell Lines through Transcriptional Mechanism

    doi: 10.1155/2012/593195

    Figure Lengend Snippet: Effect of MAPKs inhibitors on camel milk mediated induction of caspase-3 mRNA levels in HepG2 cells. HepG2 cells were pre-treated with for 2 h with JNK inhibitor, SP600125, p38 inhibitor, SB203580, and ERK inhibitor, U0126, before the addition of camel milk (10 mg/mL) for additional 6 h. Thereafter, total RNA was isolated using TRIzol reagent and the mRNA levels of Caspase-3 were quantified using RT-PCR normalized to β -actin housekeeping gene as described Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P

    Article Snippet: Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions and quantified by measuring the absorbance at 260 nm.

    Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction

    Effect of camel milk on oxidative stress markers HO-1 (a) and NQO1 (b) mRNA levels, and ROS (c) production in HepG2 cells. (a) and (b) HepG2 cells were treated for 6 h with a various concentrations of camel milk (2.5, 5, 10, and 20 mg/mL). Thereafter, total RNA was isolated using TRIzol reagent and the mRNA levels of HO-1 and NQO1 were quantified using RT-PCR normalized to β -actin housekeeping gene as described Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Camel Milk Triggers Apoptotic Signaling Pathways in Human Hepatoma HepG2 and Breast Cancer MCF7 Cell Lines through Transcriptional Mechanism

    doi: 10.1155/2012/593195

    Figure Lengend Snippet: Effect of camel milk on oxidative stress markers HO-1 (a) and NQO1 (b) mRNA levels, and ROS (c) production in HepG2 cells. (a) and (b) HepG2 cells were treated for 6 h with a various concentrations of camel milk (2.5, 5, 10, and 20 mg/mL). Thereafter, total RNA was isolated using TRIzol reagent and the mRNA levels of HO-1 and NQO1 were quantified using RT-PCR normalized to β -actin housekeeping gene as described Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P

    Article Snippet: Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions and quantified by measuring the absorbance at 260 nm.

    Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction

    Differentially expressed genes in Mkp-1 +/+ and Mkp-1 −/− mice before and following E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS (controls). Mice were euthanized after 24 h, and total RNA was isolated from the livers of four mice using Trizol for RNA-seq analyses. Volcano plots show the extent of differentially expressed gene (adjusted p value

    Journal: International Journal of Molecular Sciences

    Article Title: Dysregulation of Lipid Metabolism in Mkp-1 Deficient Mice during Gram-Negative Sepsis

    doi: 10.3390/ijms19123904

    Figure Lengend Snippet: Differentially expressed genes in Mkp-1 +/+ and Mkp-1 −/− mice before and following E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS (controls). Mice were euthanized after 24 h, and total RNA was isolated from the livers of four mice using Trizol for RNA-seq analyses. Volcano plots show the extent of differentially expressed gene (adjusted p value

    Article Snippet: Total RNA was extracted from frozen liver tissues using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA), solubilized in UltraPure RNase/DNase-free water (Thermo Fisher Scientific), and quantified by using NanoDrop ND-1000 spectrophotometer (Marshall Scientific, Hampton, NH, USA).

    Techniques: Mouse Assay, Infection, Injection, Isolation, RNA Sequencing Assay

    Liver mRNA expression levels of Cd36 in control and E. coli -infected mice. Control and E. coli -infected mice (2.5 × 10 7 CFU/g of body weight, i.v.) were euthanized 24 h post infection. Total RNA was isolated from the livers using Trizol. Cd36 mRNA levels were assessed via qRT-PCR. Expression in un-infected Mkp-1 +/+ mice was set as 1. Values represent means ± S.E. from 4–7 animals in each group. The results were analyzed by two-way ANOVA. Groups marked with distinct letters above the bars indicate significant differences ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Dysregulation of Lipid Metabolism in Mkp-1 Deficient Mice during Gram-Negative Sepsis

    doi: 10.3390/ijms19123904

    Figure Lengend Snippet: Liver mRNA expression levels of Cd36 in control and E. coli -infected mice. Control and E. coli -infected mice (2.5 × 10 7 CFU/g of body weight, i.v.) were euthanized 24 h post infection. Total RNA was isolated from the livers using Trizol. Cd36 mRNA levels were assessed via qRT-PCR. Expression in un-infected Mkp-1 +/+ mice was set as 1. Values represent means ± S.E. from 4–7 animals in each group. The results were analyzed by two-way ANOVA. Groups marked with distinct letters above the bars indicate significant differences ( p

    Article Snippet: Total RNA was extracted from frozen liver tissues using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA), solubilized in UltraPure RNase/DNase-free water (Thermo Fisher Scientific), and quantified by using NanoDrop ND-1000 spectrophotometer (Marshall Scientific, Hampton, NH, USA).

    Techniques: Expressing, Infection, Mouse Assay, Isolation, Quantitative RT-PCR

    Hepatic mRNA expression of lipogenesis genes before and after E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS. Mice were euthanized after 24 h, and total RNA was isolated from the livers using Trizol. mRNA expression for different genes was assessed based on the RNA-seq data set, or quantified via qRT-PCR. Expression in un-infected Mkp-1 +/+ mice was set as 1. Values represent means ± S.E. from 4 animals for RNA-seq and 4–7 animals for qRT-PCR in each group. ( A ) Expression levels of lipogenic regulator genes based on RNA-seq; ( B ) Expression levels of Mtor and Pparg based on qRT-PCR; ( C ) Expression levels of lipogenic genes based on RNA-seq. Values in the graphs were compared by two-way ANOVA. Groups marked with distinct letters above the bars indicate significant differences ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Dysregulation of Lipid Metabolism in Mkp-1 Deficient Mice during Gram-Negative Sepsis

    doi: 10.3390/ijms19123904

    Figure Lengend Snippet: Hepatic mRNA expression of lipogenesis genes before and after E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS. Mice were euthanized after 24 h, and total RNA was isolated from the livers using Trizol. mRNA expression for different genes was assessed based on the RNA-seq data set, or quantified via qRT-PCR. Expression in un-infected Mkp-1 +/+ mice was set as 1. Values represent means ± S.E. from 4 animals for RNA-seq and 4–7 animals for qRT-PCR in each group. ( A ) Expression levels of lipogenic regulator genes based on RNA-seq; ( B ) Expression levels of Mtor and Pparg based on qRT-PCR; ( C ) Expression levels of lipogenic genes based on RNA-seq. Values in the graphs were compared by two-way ANOVA. Groups marked with distinct letters above the bars indicate significant differences ( p

    Article Snippet: Total RNA was extracted from frozen liver tissues using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA), solubilized in UltraPure RNase/DNase-free water (Thermo Fisher Scientific), and quantified by using NanoDrop ND-1000 spectrophotometer (Marshall Scientific, Hampton, NH, USA).

    Techniques: Expressing, Infection, Mouse Assay, Injection, Isolation, RNA Sequencing Assay, Quantitative RT-PCR

    p65 inhibited E3 ligase FBW7 degradation. (A B) T24T(shp65-1) vs. T24T(nonsense) or MEF(p65−/−), MEF[p65−/−(p65)] vs. MEF(WT) cells were extracted for total RNA with Trizol reagent. RT-PCR assay was used to determine fbw7 mRNA expression. β-Actin was used as an internal control. (C) The indicated cells were pretreated with proteasome inhibitor MG132 for 10 hours, and the cells were then treated with CHX for the indicated times. Then cell extracts were subjected to Western blot, and GAPDH was used as a protein loading control.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: NF-κB p65 Overexpression Promotes Bladder Cancer Cell Migration via FBW7-Mediated Degradation of RhoGDIα Protein

    doi: 10.1016/j.neo.2017.06.002

    Figure Lengend Snippet: p65 inhibited E3 ligase FBW7 degradation. (A B) T24T(shp65-1) vs. T24T(nonsense) or MEF(p65−/−), MEF[p65−/−(p65)] vs. MEF(WT) cells were extracted for total RNA with Trizol reagent. RT-PCR assay was used to determine fbw7 mRNA expression. β-Actin was used as an internal control. (C) The indicated cells were pretreated with proteasome inhibitor MG132 for 10 hours, and the cells were then treated with CHX for the indicated times. Then cell extracts were subjected to Western blot, and GAPDH was used as a protein loading control.

    Article Snippet: Total RNA was extracted by using the TRIzol reagent (Invitrogen, Grand Island, NY) as described in the manufacturer's instructions.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

    PTEN was p65 a downstream effector and promoted FBW7 protein degradation and inhibited BC migration. (A) The indicated cell extracts were subjected to Western blot to determine protein expression of PTEN, AKT, p-AKT(Thr308), and p-AKT(Ser473). (B) The indicated cell extracts were subjected to Western blot to determine protein expression of PTEN. GAPDH was used as a protein loading control. (C) MEF(p65−/−), MEF[p65−/−(p65)] vs. MEF(WT) cells were extracted for total RNA with Trizol reagent. RT-PCR assay was used to determine pten mRNA expression. β-Actin was used as an internal control. (D) Human PTEN promoter-driven luciferase activity was evaluated in MEF(p65−/−), MEF[p65−/−(p65)] and MEF(WT) cells. The results were normalized by internal TK activity. Bars represent mean ± SD from three independent experiments. Student's t test was utilized to determine the P value. The symbol (♣) indicates a significant increase as compared with MEF(WT) cells ( ♣ P

    Journal: Neoplasia (New York, N.Y.)

    Article Title: NF-κB p65 Overexpression Promotes Bladder Cancer Cell Migration via FBW7-Mediated Degradation of RhoGDIα Protein

    doi: 10.1016/j.neo.2017.06.002

    Figure Lengend Snippet: PTEN was p65 a downstream effector and promoted FBW7 protein degradation and inhibited BC migration. (A) The indicated cell extracts were subjected to Western blot to determine protein expression of PTEN, AKT, p-AKT(Thr308), and p-AKT(Ser473). (B) The indicated cell extracts were subjected to Western blot to determine protein expression of PTEN. GAPDH was used as a protein loading control. (C) MEF(p65−/−), MEF[p65−/−(p65)] vs. MEF(WT) cells were extracted for total RNA with Trizol reagent. RT-PCR assay was used to determine pten mRNA expression. β-Actin was used as an internal control. (D) Human PTEN promoter-driven luciferase activity was evaluated in MEF(p65−/−), MEF[p65−/−(p65)] and MEF(WT) cells. The results were normalized by internal TK activity. Bars represent mean ± SD from three independent experiments. Student's t test was utilized to determine the P value. The symbol (♣) indicates a significant increase as compared with MEF(WT) cells ( ♣ P

    Article Snippet: Total RNA was extracted by using the TRIzol reagent (Invitrogen, Grand Island, NY) as described in the manufacturer's instructions.

    Techniques: Migration, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Luciferase, Activity Assay

    p65 promoted degradation of RhoGDIα protein. (A B) T24T(shp65-1), T24T(shp65-2) vs. T24T(nonsense) or MEF(p65−/−), MEF[p65−/−(p65)] vs. MEF(WT) cells were extracted for total RNA with Trizol reagent. RT-PCR assay was used to determine RhoGDiα mRNA expression. β-Actin was used as an internal control. (C) The indicated cells were pretreated with proteasome inhibitor MG132 for 10 hours and were then followed by treatment of cells with CHX for the indicated times. The cell extracts were subjected to Western blot, and GAPDH was used as a protein loading control.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: NF-κB p65 Overexpression Promotes Bladder Cancer Cell Migration via FBW7-Mediated Degradation of RhoGDIα Protein

    doi: 10.1016/j.neo.2017.06.002

    Figure Lengend Snippet: p65 promoted degradation of RhoGDIα protein. (A B) T24T(shp65-1), T24T(shp65-2) vs. T24T(nonsense) or MEF(p65−/−), MEF[p65−/−(p65)] vs. MEF(WT) cells were extracted for total RNA with Trizol reagent. RT-PCR assay was used to determine RhoGDiα mRNA expression. β-Actin was used as an internal control. (C) The indicated cells were pretreated with proteasome inhibitor MG132 for 10 hours and were then followed by treatment of cells with CHX for the indicated times. The cell extracts were subjected to Western blot, and GAPDH was used as a protein loading control.

    Article Snippet: Total RNA was extracted by using the TRIzol reagent (Invitrogen, Grand Island, NY) as described in the manufacturer's instructions.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

    Relative mRNA expression of estrogen-relative genes in uterus from immature female rats exposed to Ginkgo biloba extract (EGb761) alone (A) or the combination of EGb761 and 17β-estradiol (E2) or tamoxifen (TM) (B). The total RNA was extracted using TRIzol in liver obtained from the immature female rats exposed to respective compounds. The mRNA levels of CaBP-9 were measured using RT-PCR along with cytochrome c Oxidase I (CO1) mRNA as the internal standard. The PCR product was identified using a gel documentation and quantified by ImageJ 1.43u. The results are expressed as mean ± SD of three separate experiments for each group. Values significantly different from the control are indicated by an asterisk (*p

    Journal: Environmental Health and Toxicology

    Article Title: Ginkgo biloba extract (EGb761) did not express estrogenic activity in an immature rat uterotrophic assay

    doi: 10.5620/eht.e2018016

    Figure Lengend Snippet: Relative mRNA expression of estrogen-relative genes in uterus from immature female rats exposed to Ginkgo biloba extract (EGb761) alone (A) or the combination of EGb761 and 17β-estradiol (E2) or tamoxifen (TM) (B). The total RNA was extracted using TRIzol in liver obtained from the immature female rats exposed to respective compounds. The mRNA levels of CaBP-9 were measured using RT-PCR along with cytochrome c Oxidase I (CO1) mRNA as the internal standard. The PCR product was identified using a gel documentation and quantified by ImageJ 1.43u. The results are expressed as mean ± SD of three separate experiments for each group. Values significantly different from the control are indicated by an asterisk (*p

    Article Snippet: Total RNA was extracted from uteruses and livers using Trizol Reagent (Gibco BRL, Grand Island, NY, USA) according to the manufacturer’s protocol and stored at -80°C until needed.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Relative mRNA expression of estrogen-relative genes in liver from immature female rats exposed to Ginkgo biloba extract (EGb761) alone (A) or the combination of EGb761 and 17β-estradiol (E2) or tamoxifen (TM) (B). The total RNA was extracted using TRIzol in liver obtained from the immature female rats exposed to respective compounds. The mRNA levels (IGFBP) were measured using RT-PCR along with cytochrome c Oxidase I (CO1) mRNA as the internal standard. The PCR product was identified using a gel documentation and quantified by ImageJ 1.43u. The results are expressed as mean ± SD of three separate experiments for each group. Values significantly different from the control are indicated by an asterisk (**p

    Journal: Environmental Health and Toxicology

    Article Title: Ginkgo biloba extract (EGb761) did not express estrogenic activity in an immature rat uterotrophic assay

    doi: 10.5620/eht.e2018016

    Figure Lengend Snippet: Relative mRNA expression of estrogen-relative genes in liver from immature female rats exposed to Ginkgo biloba extract (EGb761) alone (A) or the combination of EGb761 and 17β-estradiol (E2) or tamoxifen (TM) (B). The total RNA was extracted using TRIzol in liver obtained from the immature female rats exposed to respective compounds. The mRNA levels (IGFBP) were measured using RT-PCR along with cytochrome c Oxidase I (CO1) mRNA as the internal standard. The PCR product was identified using a gel documentation and quantified by ImageJ 1.43u. The results are expressed as mean ± SD of three separate experiments for each group. Values significantly different from the control are indicated by an asterisk (**p

    Article Snippet: Total RNA was extracted from uteruses and livers using Trizol Reagent (Gibco BRL, Grand Island, NY, USA) according to the manufacturer’s protocol and stored at -80°C until needed.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Renal expression of AT1 (a) and TGF- β 1 (b) in ZL and ZDF rats, and levels of AT1 (c) and TGF- β 1 (d) mRNAs, and TGF- β protein (e) in rat mesangial cells. In cell culture, SO (SO10: 10; SO20: 20 and SO40: 40 μ g/ml), MA (25 μ M) or telmisartan (Tel. 10 μ M) was added 1 h prior to treatment with angiotensin II (10 −6 M), followed by twenty-four hours incubation. Total RNA was extracted from renal tissues and the mesangial cells using TRIzol, respectively. The relative levels of specific mRNAs were determined by RT–PCR. Results were normalized to β -actin. In a parallel experiment, the medium was collected after mesangial cells were treated with the test agents in combination with angiotensin II. TGF- β protein level was determined by commercial ELISA kit according to the manufacturer's instructions. All values are means ± SEM ( n = 5, each group in vivo ; n = 3, each group in vitro ). * P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: The Ayurvedic Medicine Salacia oblonga Attenuates Diabetic Renal Fibrosis in Rats: Suppression of Angiotensin II/AT1 Signaling

    doi: 10.1093/ecam/nep095

    Figure Lengend Snippet: Renal expression of AT1 (a) and TGF- β 1 (b) in ZL and ZDF rats, and levels of AT1 (c) and TGF- β 1 (d) mRNAs, and TGF- β protein (e) in rat mesangial cells. In cell culture, SO (SO10: 10; SO20: 20 and SO40: 40 μ g/ml), MA (25 μ M) or telmisartan (Tel. 10 μ M) was added 1 h prior to treatment with angiotensin II (10 −6 M), followed by twenty-four hours incubation. Total RNA was extracted from renal tissues and the mesangial cells using TRIzol, respectively. The relative levels of specific mRNAs were determined by RT–PCR. Results were normalized to β -actin. In a parallel experiment, the medium was collected after mesangial cells were treated with the test agents in combination with angiotensin II. TGF- β protein level was determined by commercial ELISA kit according to the manufacturer's instructions. All values are means ± SEM ( n = 5, each group in vivo ; n = 3, each group in vitro ). * P

    Article Snippet: Total RNA was prepared from the kidneys of individual rats or rat mesangial cells using TRIzol reagent (Invitrogen, Australia and Invitrogen, China).

    Techniques: Expressing, Cell Culture, Incubation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, In Vivo, In Vitro

    Renal expression of PAI-1 (a), uPA (b) mRNAs, and the ratio of uPA to PAI-1 (c) in ZL and ZDF rats, and levels of PAI-1 (d) and uPA (e) mRNAs, and the ratio of uPA to PAI-1 (f) in rat mesangial cells. In cell culture, SO (SO10: 10 and SO40: 40 μ g/ml), MA (25 μ M) or Tel (10 μ M) was added 1 h prior to treatment with angiotensin II (10 −6 M), followed by 24-h incubation. Total RNA was extracted from renal tissues and the mesangial cells using TRIzol, respectively. The relative levels of specific mRNAs were determined by RT–PCR. Results were normalized to β -actin. All values are means ± SEM ( n = 5, each group in vivo ; n = 3, each group in vitro ). * P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: The Ayurvedic Medicine Salacia oblonga Attenuates Diabetic Renal Fibrosis in Rats: Suppression of Angiotensin II/AT1 Signaling

    doi: 10.1093/ecam/nep095

    Figure Lengend Snippet: Renal expression of PAI-1 (a), uPA (b) mRNAs, and the ratio of uPA to PAI-1 (c) in ZL and ZDF rats, and levels of PAI-1 (d) and uPA (e) mRNAs, and the ratio of uPA to PAI-1 (f) in rat mesangial cells. In cell culture, SO (SO10: 10 and SO40: 40 μ g/ml), MA (25 μ M) or Tel (10 μ M) was added 1 h prior to treatment with angiotensin II (10 −6 M), followed by 24-h incubation. Total RNA was extracted from renal tissues and the mesangial cells using TRIzol, respectively. The relative levels of specific mRNAs were determined by RT–PCR. Results were normalized to β -actin. All values are means ± SEM ( n = 5, each group in vivo ; n = 3, each group in vitro ). * P

    Article Snippet: Total RNA was prepared from the kidneys of individual rats or rat mesangial cells using TRIzol reagent (Invitrogen, Australia and Invitrogen, China).

    Techniques: Expressing, Cell Culture, Incubation, Reverse Transcription Polymerase Chain Reaction, In Vivo, In Vitro

    Expression of collagen (Col) I (a), Col IV (b), fibronectin (c) mRNAs and fibronectin protein (d) in rat mesangial cells. SO (SO10: 10; SO20: 20 and SO40: 40 μ g/ml), MA (25 μ M) or telmisartan (Tel. 10 μ M) was added 1 h before mesangial cells were treated with angiotensin II (10 −6 M). Twenty-four hours later total RNA was extracted from the mesangial cells using TRIzol. The relative levels of specific mRNAs were determined by RT-PCR. Results were normalized to β -actin. In a parallel experiment, the medium was collected after mesangial cells were treated with the test agents in combination with angiotensin II. fibronectin protein was determined by commercial ELISA kit according to the manufacturer's instructions. All values are means ± SEM ( n = 3, each group). * P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: The Ayurvedic Medicine Salacia oblonga Attenuates Diabetic Renal Fibrosis in Rats: Suppression of Angiotensin II/AT1 Signaling

    doi: 10.1093/ecam/nep095

    Figure Lengend Snippet: Expression of collagen (Col) I (a), Col IV (b), fibronectin (c) mRNAs and fibronectin protein (d) in rat mesangial cells. SO (SO10: 10; SO20: 20 and SO40: 40 μ g/ml), MA (25 μ M) or telmisartan (Tel. 10 μ M) was added 1 h before mesangial cells were treated with angiotensin II (10 −6 M). Twenty-four hours later total RNA was extracted from the mesangial cells using TRIzol. The relative levels of specific mRNAs were determined by RT-PCR. Results were normalized to β -actin. In a parallel experiment, the medium was collected after mesangial cells were treated with the test agents in combination with angiotensin II. fibronectin protein was determined by commercial ELISA kit according to the manufacturer's instructions. All values are means ± SEM ( n = 3, each group). * P

    Article Snippet: Total RNA was prepared from the kidneys of individual rats or rat mesangial cells using TRIzol reagent (Invitrogen, Australia and Invitrogen, China).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    HuR antagonizes the downregulation of COX-2 expression caused by miR-16 in hepatoma cell lines. WRL68 cell extracts (500 µg per lane) were immunoprecipitated with HuR or IgG antibodies. Bound RNA was harvested with TRIzol reagent, reverse transcriptased, and PCR amplified with COX-2 ( A ) or miR-16 primers ( B ). PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. The abundance of the transcripts present in WRL68 cells after HuR immunoprecipitation was assessed, and fold differences were plotted. Input, total mRNA in cell extract; unbound, unbound mRNA after immunoprecipitation with HuR antibody; bound, bound mRNA after immunoprecipitation with HuR antibody; and control, bound mRNA after immunoprecipitation with IgG antiboby. ( C–D ) WRL68 and Hep3B cell lines were transfected with miR-16 or In-miR-16 (50 nM). COX-2 and HuR protein levels were analyzed by Western Blot. ( E–F ) WRL68 and Hep3B cell lines were cotransfected with miR-16 (50 nM) and pcDNA3-HuR-GFP expression vector (4 µg). COX-2 and HuR protein levels were analyzed by Western Blot. Data are reported as means±SD of three independent experiments. *p

    Journal: PLoS ONE

    Article Title: Cyclooxygenase-2 Is a Target of MicroRNA-16 in Human Hepatoma Cells

    doi: 10.1371/journal.pone.0050935

    Figure Lengend Snippet: HuR antagonizes the downregulation of COX-2 expression caused by miR-16 in hepatoma cell lines. WRL68 cell extracts (500 µg per lane) were immunoprecipitated with HuR or IgG antibodies. Bound RNA was harvested with TRIzol reagent, reverse transcriptased, and PCR amplified with COX-2 ( A ) or miR-16 primers ( B ). PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. The abundance of the transcripts present in WRL68 cells after HuR immunoprecipitation was assessed, and fold differences were plotted. Input, total mRNA in cell extract; unbound, unbound mRNA after immunoprecipitation with HuR antibody; bound, bound mRNA after immunoprecipitation with HuR antibody; and control, bound mRNA after immunoprecipitation with IgG antiboby. ( C–D ) WRL68 and Hep3B cell lines were transfected with miR-16 or In-miR-16 (50 nM). COX-2 and HuR protein levels were analyzed by Western Blot. ( E–F ) WRL68 and Hep3B cell lines were cotransfected with miR-16 (50 nM) and pcDNA3-HuR-GFP expression vector (4 µg). COX-2 and HuR protein levels were analyzed by Western Blot. Data are reported as means±SD of three independent experiments. *p

    Article Snippet: Total RNA from HCC cells or human biopsies was extracted by using TRIzol reagent (Invitrogen, Grand Island, NY, USA).

    Techniques: Expressing, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Electrophoresis, Staining, Transfection, Western Blot, Plasmid Preparation

    miR-16 binds COX-2 mRNA and inhibits its translation. ( A ) WRL68 cell extracts (500 µg per lane) were immunoprecipitated with Ago-2 or IgG antibodies. Bound RNA was harvested with TRIzol reagent, reverse transcriptased, and PCR amplified with COX-2 primers. PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. The presence of COX-2 mRNA in WRL68 cell transfected with miR-16 or Lipofectamine after Ago2 immunoprecipitation was assessed, and fold differences were plotted. Input, total mRNA in cell extract; and control, bound mRNA after immunoprecipitation with IgG antiboby. ( B ) Scheme of pGL3-empty, pGL3-seed and pGL3-mut reporter vectors. In pGL3-seed, the putative binding site of miR-16 on COX-2 mRNA 3′-UTR region (as detected by RNAhybrid software) was introduced downstream luciferase gene. In pGL3-mut this region was mutated in order to avoid the binding between miR-16 and Luc mRNA. ( C–D ) A luciferase assay was carried out on HuH-7 and HepG2 cell lines using pGL3-seed and pGL3-mut reporter vectors. Firefly luciferase activity was evaluated 48 h after co-transfection with pGL3-empty/seed/mut (750 ng), miR-16 (50 nM), In-miR-16 (50 nM) and miR-NC (50 nM) as indicated. Data were normalized against renilla luciferase activity (all samples were co-transfected with 50 ng pRL vector and refer to the positive control, pGL3 empty vector). Data are reported as means±SD of three independent experiments. *p

    Journal: PLoS ONE

    Article Title: Cyclooxygenase-2 Is a Target of MicroRNA-16 in Human Hepatoma Cells

    doi: 10.1371/journal.pone.0050935

    Figure Lengend Snippet: miR-16 binds COX-2 mRNA and inhibits its translation. ( A ) WRL68 cell extracts (500 µg per lane) were immunoprecipitated with Ago-2 or IgG antibodies. Bound RNA was harvested with TRIzol reagent, reverse transcriptased, and PCR amplified with COX-2 primers. PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. The presence of COX-2 mRNA in WRL68 cell transfected with miR-16 or Lipofectamine after Ago2 immunoprecipitation was assessed, and fold differences were plotted. Input, total mRNA in cell extract; and control, bound mRNA after immunoprecipitation with IgG antiboby. ( B ) Scheme of pGL3-empty, pGL3-seed and pGL3-mut reporter vectors. In pGL3-seed, the putative binding site of miR-16 on COX-2 mRNA 3′-UTR region (as detected by RNAhybrid software) was introduced downstream luciferase gene. In pGL3-mut this region was mutated in order to avoid the binding between miR-16 and Luc mRNA. ( C–D ) A luciferase assay was carried out on HuH-7 and HepG2 cell lines using pGL3-seed and pGL3-mut reporter vectors. Firefly luciferase activity was evaluated 48 h after co-transfection with pGL3-empty/seed/mut (750 ng), miR-16 (50 nM), In-miR-16 (50 nM) and miR-NC (50 nM) as indicated. Data were normalized against renilla luciferase activity (all samples were co-transfected with 50 ng pRL vector and refer to the positive control, pGL3 empty vector). Data are reported as means±SD of three independent experiments. *p

    Article Snippet: Total RNA from HCC cells or human biopsies was extracted by using TRIzol reagent (Invitrogen, Grand Island, NY, USA).

    Techniques: Immunoprecipitation, Polymerase Chain Reaction, Amplification, Electrophoresis, Staining, Transfection, Binding Assay, Software, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Positive Control

    Effect of miR-16 on COX-2 mRNA and protein stability. Hep3B cells were transfected with 50 nM miR-16 or miR-NC, or 30 nM siCOX-2. 5 µg/ml actinomycin-D (Act D) or 10 µg/ml cycloheximide (CHX) were added after transfection. ( A–B ) COX-2 protein was analyzed by Western blot at different time after actinomycin-D treatment. Corresponding densitometry analysis is shown and the relative expression of each sample is related to sample at 0 h as 1. ( C ) mRNA COX-2 levels were analyzed by real time PCR. COX-2 mRNA amounts were calculated as relative expression and normalized to the expression of 36b4 mRNA. Values represent fold change relative to sample at 0 h. ( D–E ) COX-2 protein levels were analyzed by Western blot in the presence or absence of cycloheximide. Corresponding densitometric analysis is shown and the relative expression of each sample is related to the value at 0 h as 1. F) Hep3B cells were transfected with 50 nM miR-16, miR-16 inhibitor (In-miR-16) or lipofectamine and permeabilized with digitonine to obtain soluble and pellet fractions enriched in PB as described in Methods. RNA was isolated from each fraction with Trizol reagent, reverse transcriptased, and PCR amplified with COX-2, Xrn1 and actin primers. Input, RNA extracted from cells prior to fractionation. PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. G) The presence of COX-2 mRNA in soluble and PB fractions was assessed and fold differences were plotted. Data are reported as means±SD of three independent experiments. **p

    Journal: PLoS ONE

    Article Title: Cyclooxygenase-2 Is a Target of MicroRNA-16 in Human Hepatoma Cells

    doi: 10.1371/journal.pone.0050935

    Figure Lengend Snippet: Effect of miR-16 on COX-2 mRNA and protein stability. Hep3B cells were transfected with 50 nM miR-16 or miR-NC, or 30 nM siCOX-2. 5 µg/ml actinomycin-D (Act D) or 10 µg/ml cycloheximide (CHX) were added after transfection. ( A–B ) COX-2 protein was analyzed by Western blot at different time after actinomycin-D treatment. Corresponding densitometry analysis is shown and the relative expression of each sample is related to sample at 0 h as 1. ( C ) mRNA COX-2 levels were analyzed by real time PCR. COX-2 mRNA amounts were calculated as relative expression and normalized to the expression of 36b4 mRNA. Values represent fold change relative to sample at 0 h. ( D–E ) COX-2 protein levels were analyzed by Western blot in the presence or absence of cycloheximide. Corresponding densitometric analysis is shown and the relative expression of each sample is related to the value at 0 h as 1. F) Hep3B cells were transfected with 50 nM miR-16, miR-16 inhibitor (In-miR-16) or lipofectamine and permeabilized with digitonine to obtain soluble and pellet fractions enriched in PB as described in Methods. RNA was isolated from each fraction with Trizol reagent, reverse transcriptased, and PCR amplified with COX-2, Xrn1 and actin primers. Input, RNA extracted from cells prior to fractionation. PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. G) The presence of COX-2 mRNA in soluble and PB fractions was assessed and fold differences were plotted. Data are reported as means±SD of three independent experiments. **p

    Article Snippet: Total RNA from HCC cells or human biopsies was extracted by using TRIzol reagent (Invitrogen, Grand Island, NY, USA).

    Techniques: Transfection, Activated Clotting Time Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Amplification, Fractionation, Electrophoresis, Staining

    Analysis of HO1 mRNA abundance and protein levels in tissues of experimental mice. HO1 transcript abundance was measured in skeletal muscle (A,B) , neuronal tissues (C) , and liver (D) (left-hand) by real-time RT-PCR as described in M M. The histograms display HO1 mRNA levels after normalization to 18S ribosomal RNA levels. Each column represents the mean ± SD of three biological amplification reactions. RNA from each mouse was extracted separately using TRIZOL reagent, reverse-transcribed and amplified as described in M M; therefore, 18 cDNA samples (biological triplicates for each condition) were finally used in the analysis of gene expression in each tissue. The same capital and small letter indicates groups between which statistically significant differences were found at P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Mice Overexpressing Both Non-Mutated Human SOD1 and Mutated SOD1G93A Genes: A Competent Experimental Model for Studying Iron Metabolism in Amyotrophic Lateral Sclerosis

    doi: 10.3389/fnmol.2015.00082

    Figure Lengend Snippet: Analysis of HO1 mRNA abundance and protein levels in tissues of experimental mice. HO1 transcript abundance was measured in skeletal muscle (A,B) , neuronal tissues (C) , and liver (D) (left-hand) by real-time RT-PCR as described in M M. The histograms display HO1 mRNA levels after normalization to 18S ribosomal RNA levels. Each column represents the mean ± SD of three biological amplification reactions. RNA from each mouse was extracted separately using TRIZOL reagent, reverse-transcribed and amplified as described in M M; therefore, 18 cDNA samples (biological triplicates for each condition) were finally used in the analysis of gene expression in each tissue. The same capital and small letter indicates groups between which statistically significant differences were found at P

    Article Snippet: Total RNA was extracted using the TRIzol reagent (Invitrogen).

    Techniques: Mouse Assay, Quantitative RT-PCR, Amplification, Expressing

    Analysis of TfR1 mRNA abundance in tissues of experimental mice. TfR1 mRNA abundance in skeletal muscles (A,B) , spinal cord (C), and liver (D) was measured by real-time RT-PCR as described under “Materials and methods.” The histograms display TfR1 mRNA levels after normalization to 18S ribosomal RNA levels. Each column represents the mean ± SD of three biological amplification reactions. RNA from each mouse was extracted separately using TRIZOL reagent, reverse-transcribed and amplified as described in M M; therefore, 18 cDNA samples (biological triplicates for each condition) were finally used in the analysis of gene expression in each tissue. ∗ , significant difference versus wild-type in the group of symptomatic, 4-month-old mice ( P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Mice Overexpressing Both Non-Mutated Human SOD1 and Mutated SOD1G93A Genes: A Competent Experimental Model for Studying Iron Metabolism in Amyotrophic Lateral Sclerosis

    doi: 10.3389/fnmol.2015.00082

    Figure Lengend Snippet: Analysis of TfR1 mRNA abundance in tissues of experimental mice. TfR1 mRNA abundance in skeletal muscles (A,B) , spinal cord (C), and liver (D) was measured by real-time RT-PCR as described under “Materials and methods.” The histograms display TfR1 mRNA levels after normalization to 18S ribosomal RNA levels. Each column represents the mean ± SD of three biological amplification reactions. RNA from each mouse was extracted separately using TRIZOL reagent, reverse-transcribed and amplified as described in M M; therefore, 18 cDNA samples (biological triplicates for each condition) were finally used in the analysis of gene expression in each tissue. ∗ , significant difference versus wild-type in the group of symptomatic, 4-month-old mice ( P

    Article Snippet: Total RNA was extracted using the TRIzol reagent (Invitrogen).

    Techniques: Mouse Assay, Quantitative RT-PCR, Amplification, Expressing

    MLN51 expression in dendritic cells (DCs) and its effect on the proliferation of immature DCs. (a) MLN51 expression is upregulated only in immature bone marrow-derived dendritic cells (BmDCs) in contrast with bone marrow progenitor cells or mature BmDCs. Total RNA was extracted from each sample with Trizol reagent. RT-PCR was performed with mouse MLN51 primers. (b) BC-1 cells (10 6 ) were transfected with 4 μg of mMLN51 siRNA (siRNA(+)) or control siRNA (siRNA(-)). The transfected cells were harvested every day and assessed for their proliferation over a period of 4 days. The results are means ± SD obtained from single experiments performed in triplicate cultures. (c) Total RNA was extracted each day from the transfected BC-1 cell cultures. mMLN51 was assessed in each sample by RT-PCR as described previously with primers. Results in (a) and (c) are representative of three separate experiments.

    Journal: Arthritis Research & Therapy

    Article Title: MLN51 and GM-CSF involvement in the proliferation of fibroblast-like synoviocytes in the pathogenesis of rheumatoid arthritis

    doi: 10.1186/ar2079

    Figure Lengend Snippet: MLN51 expression in dendritic cells (DCs) and its effect on the proliferation of immature DCs. (a) MLN51 expression is upregulated only in immature bone marrow-derived dendritic cells (BmDCs) in contrast with bone marrow progenitor cells or mature BmDCs. Total RNA was extracted from each sample with Trizol reagent. RT-PCR was performed with mouse MLN51 primers. (b) BC-1 cells (10 6 ) were transfected with 4 μg of mMLN51 siRNA (siRNA(+)) or control siRNA (siRNA(-)). The transfected cells were harvested every day and assessed for their proliferation over a period of 4 days. The results are means ± SD obtained from single experiments performed in triplicate cultures. (c) Total RNA was extracted each day from the transfected BC-1 cell cultures. mMLN51 was assessed in each sample by RT-PCR as described previously with primers. Results in (a) and (c) are representative of three separate experiments.

    Article Snippet: Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA) and purified by using the RNeasy total RNA isolation kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer's instructions.

    Techniques: Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Transfection

    MLN51 expression is upregulated in rheumatoid arthritis FLSs compared with osteoarthritis FLSs. (a) Total RNA sample (1 μg) was extracted from three rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) and one osteoarthritis (OA) FLS with Trizol reagent. RT-PCR was performed with 5 ng of cDNA as a template and MLN51 -specific or GAPDH-specific primers. The band for OA FLSs resulted from one (2–43) of the three OA FLSs. (b) Western blot analysis of MLN51 in FLS samples. RA FLSs (2–18, 2–36 and 2–38) and OA FLSs (2–43, 2–46 and 2–47) isolated from each patient were seeded at 5 × 10 4 cells per well in a six-well plate. FLSs grown in high-glucose DMEM supplemented with 10% FBS were harvested, separated by 10% SDS-PAGE, transferred to a nitrocellulose membrane and then proved with anti-hMLN51 rabbit serum (1:1,000 dilution) and horseradish peroxidase-conjugated anti-rabbit IgG (1:5,000 dilution). Data in (a) and (b) are representative of three or four separate experiments.

    Journal: Arthritis Research & Therapy

    Article Title: MLN51 and GM-CSF involvement in the proliferation of fibroblast-like synoviocytes in the pathogenesis of rheumatoid arthritis

    doi: 10.1186/ar2079

    Figure Lengend Snippet: MLN51 expression is upregulated in rheumatoid arthritis FLSs compared with osteoarthritis FLSs. (a) Total RNA sample (1 μg) was extracted from three rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) and one osteoarthritis (OA) FLS with Trizol reagent. RT-PCR was performed with 5 ng of cDNA as a template and MLN51 -specific or GAPDH-specific primers. The band for OA FLSs resulted from one (2–43) of the three OA FLSs. (b) Western blot analysis of MLN51 in FLS samples. RA FLSs (2–18, 2–36 and 2–38) and OA FLSs (2–43, 2–46 and 2–47) isolated from each patient were seeded at 5 × 10 4 cells per well in a six-well plate. FLSs grown in high-glucose DMEM supplemented with 10% FBS were harvested, separated by 10% SDS-PAGE, transferred to a nitrocellulose membrane and then proved with anti-hMLN51 rabbit serum (1:1,000 dilution) and horseradish peroxidase-conjugated anti-rabbit IgG (1:5,000 dilution). Data in (a) and (b) are representative of three or four separate experiments.

    Article Snippet: Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA) and purified by using the RNeasy total RNA isolation kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer's instructions.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Isolation, SDS Page

    Effect of DOX and DOX-Vit D on the oxidative stress. MG63 cells were treated for 24 h with 10 µM DOX and DOX-Vit D. Thereafter, total RNA was isolated using TRIzol reagent, and the mRNA levels of ( A ) NQO-1 and ( B ) HO-1 were quantified using real time-PCR and normalized to a β-actin housekeeping gene. ( C ) MG63 cells were treated for 24 h with 10 µM DOX and DOX-Vit D then, cells were incubated with DCF-DA (10 μM) for 1 h. DCF formation was measured fluorometrically using excitation/emission wavelengths of 484/535 nm. The results are presented as the mean ± SEM ( n = 6). + p

    Journal: Pharmaceutics

    Article Title: DOX-Vit D, a Novel Doxorubicin Delivery Approach, Inhibits Human Osteosarcoma Cell Proliferation by Inducing Apoptosis While Inhibiting Akt and mTOR Signaling Pathways

    doi: 10.3390/pharmaceutics10030144

    Figure Lengend Snippet: Effect of DOX and DOX-Vit D on the oxidative stress. MG63 cells were treated for 24 h with 10 µM DOX and DOX-Vit D. Thereafter, total RNA was isolated using TRIzol reagent, and the mRNA levels of ( A ) NQO-1 and ( B ) HO-1 were quantified using real time-PCR and normalized to a β-actin housekeeping gene. ( C ) MG63 cells were treated for 24 h with 10 µM DOX and DOX-Vit D then, cells were incubated with DCF-DA (10 μM) for 1 h. DCF formation was measured fluorometrically using excitation/emission wavelengths of 484/535 nm. The results are presented as the mean ± SEM ( n = 6). + p

    Article Snippet: Total RNA was extracted using TRIzol reagent (Invitrogen® , Carlsbad, CA, USA) as described previously [ ].

    Techniques: Isolation, Real-time Polymerase Chain Reaction, Incubation

    Effect of DOX and DOX-Vit D on the expression of DR-4. MG63 cells were treated for 24 h with 10 µM DOX and DOX-Vit D. Thereafter, total RNA was isolated using TRIzol reagent, and the mRNA level of DR-4 was quantified using real time-PCR and normalized to a β-actin housekeeping gene. The results are presented as the mean ± SEM ( n = 6). + p

    Journal: Pharmaceutics

    Article Title: DOX-Vit D, a Novel Doxorubicin Delivery Approach, Inhibits Human Osteosarcoma Cell Proliferation by Inducing Apoptosis While Inhibiting Akt and mTOR Signaling Pathways

    doi: 10.3390/pharmaceutics10030144

    Figure Lengend Snippet: Effect of DOX and DOX-Vit D on the expression of DR-4. MG63 cells were treated for 24 h with 10 µM DOX and DOX-Vit D. Thereafter, total RNA was isolated using TRIzol reagent, and the mRNA level of DR-4 was quantified using real time-PCR and normalized to a β-actin housekeeping gene. The results are presented as the mean ± SEM ( n = 6). + p

    Article Snippet: Total RNA was extracted using TRIzol reagent (Invitrogen® , Carlsbad, CA, USA) as described previously [ ].

    Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction

    Effect of DOX and DOX-Vit D on proapoptotic genes. MG63 cells were treated for 24 h with 10 µM DOX and DOX-Vit D. Thereafter, total RNA was isolated using TRIzol reagent, and the mRNA levels of ( A ) Caspase-3, ( B ) p53 and ( C ) BCLxs were quantified using real time-PCR and normalized to a β-actin housekeeping gene. The results are presented as the mean ± SEM ( n = 6). + p

    Journal: Pharmaceutics

    Article Title: DOX-Vit D, a Novel Doxorubicin Delivery Approach, Inhibits Human Osteosarcoma Cell Proliferation by Inducing Apoptosis While Inhibiting Akt and mTOR Signaling Pathways

    doi: 10.3390/pharmaceutics10030144

    Figure Lengend Snippet: Effect of DOX and DOX-Vit D on proapoptotic genes. MG63 cells were treated for 24 h with 10 µM DOX and DOX-Vit D. Thereafter, total RNA was isolated using TRIzol reagent, and the mRNA levels of ( A ) Caspase-3, ( B ) p53 and ( C ) BCLxs were quantified using real time-PCR and normalized to a β-actin housekeeping gene. The results are presented as the mean ± SEM ( n = 6). + p

    Article Snippet: Total RNA was extracted using TRIzol reagent (Invitrogen® , Carlsbad, CA, USA) as described previously [ ].

    Techniques: Isolation, Real-time Polymerase Chain Reaction

    ATG7 Overexpression Decreased FOXO1 mRNA Stabilization by Regulating Its mRNA 3′ UTR Activity (A) UMUC3(shATG7#1), UMUC3(shATG7#2), and UMUC3(Nonsense) cells were cultured in 6-well plates until the cell density reached 70%–80%. Following synchronization for 12 hr, the medium was then replaced with 10% FBS DMEM for another 12 hr. Then the cells were extracted for total RNA with TRIzol reagent. RT-PCR was used to determine FOXO1 mRNA expression, and β-actin was used as an internal control. (B) The human FOXO1 promoter-driven luciferase reporter was used to evaluate its promoter transcription activity in the indicated transfectants. The results were normalized by internal TK activity. (C) UMUC3(shATG7#1), UMUC3(shATG7#2), and UMUC3(Nonsense) cells were seeded into 6-well plates. After synchronization, the indicated cells were treated with Act D for the indicated times. Total RNA was then isolated and subjected to RT-PCR analysis for mRNA levels of FOXO1 , and β-actin was used as an internal control. (D) The indicated cell extracts were subjected to western blot for determination of NCL, AUF1, and HuR protein expression. GAPDH was used as a protein loading control. (E) NCL knockdown constructs were stably transfected into UMUC3 cells. The knockdown efficiency of NCL protein and the expression of FOXO1 and p27 were evaluated by western blotting. GAPDH was used as a protein loading control. (F) The pMIR- FOXO1 3′ UTR mRNA reporter was transiently transfected into the indicated cells, and the luciferase activity of each transfectant was evaluated. The luciferase activity is presented as relative to nonsense transfectant, normalized by using pRL-TK as an internal control. The bars show mean ± SD from three independent experiments. The asterisk indicates a significant increase in UMUC3(shATG7) in comparison with nonsense transfectant (*p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: ATG7 Overexpression Is Crucial for Tumorigenic Growth of Bladder Cancer In Vitro and In Vivo by Targeting the ETS2/miRNA196b/FOXO1/p27 Axis

    doi: 10.1016/j.omtn.2017.04.012

    Figure Lengend Snippet: ATG7 Overexpression Decreased FOXO1 mRNA Stabilization by Regulating Its mRNA 3′ UTR Activity (A) UMUC3(shATG7#1), UMUC3(shATG7#2), and UMUC3(Nonsense) cells were cultured in 6-well plates until the cell density reached 70%–80%. Following synchronization for 12 hr, the medium was then replaced with 10% FBS DMEM for another 12 hr. Then the cells were extracted for total RNA with TRIzol reagent. RT-PCR was used to determine FOXO1 mRNA expression, and β-actin was used as an internal control. (B) The human FOXO1 promoter-driven luciferase reporter was used to evaluate its promoter transcription activity in the indicated transfectants. The results were normalized by internal TK activity. (C) UMUC3(shATG7#1), UMUC3(shATG7#2), and UMUC3(Nonsense) cells were seeded into 6-well plates. After synchronization, the indicated cells were treated with Act D for the indicated times. Total RNA was then isolated and subjected to RT-PCR analysis for mRNA levels of FOXO1 , and β-actin was used as an internal control. (D) The indicated cell extracts were subjected to western blot for determination of NCL, AUF1, and HuR protein expression. GAPDH was used as a protein loading control. (E) NCL knockdown constructs were stably transfected into UMUC3 cells. The knockdown efficiency of NCL protein and the expression of FOXO1 and p27 were evaluated by western blotting. GAPDH was used as a protein loading control. (F) The pMIR- FOXO1 3′ UTR mRNA reporter was transiently transfected into the indicated cells, and the luciferase activity of each transfectant was evaluated. The luciferase activity is presented as relative to nonsense transfectant, normalized by using pRL-TK as an internal control. The bars show mean ± SD from three independent experiments. The asterisk indicates a significant increase in UMUC3(shATG7) in comparison with nonsense transfectant (*p

    Article Snippet: Total RNA was extracted using TRIzol reagent (Invitrogen) as described in the manufacturer’s instructions.

    Techniques: Over Expression, Activity Assay, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing, Luciferase, Activated Clotting Time Assay, Isolation, Western Blot, Construct, Stable Transfection, Transfection

    ATG7 Overexpression Inhibited p27 mRNA Transcription in Human BC Cells (A) UMUC3(shATG7#1), UMUC3(shATG7#2), and UMUC3(Nonsense) cells were cultured in 6-well plates until the cell density reached 70%–80%. Following synchronization for 12 hr, the medium was replaced with 10% FBS DMEM for another 12 hr. Then the cells were extracted for total RNA with TRIzol reagent. RT-PCR was used to determine p27 mRNA expression, whereas β-actin was used as an internal control. (B) Schematic representation of the p27 promoter region p27 KPNI and its deletion fragment SACII . (C) p27 promoter transcription activity was evaluated by using the two p27 promoter-driven luciferase reporters shown in (B). The results were normalized by internal TK activity, and the bars show mean ± SD from three independent experiments. The asterisk indicates a significant increase in comparison with vector control transfectant (*p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: ATG7 Overexpression Is Crucial for Tumorigenic Growth of Bladder Cancer In Vitro and In Vivo by Targeting the ETS2/miRNA196b/FOXO1/p27 Axis

    doi: 10.1016/j.omtn.2017.04.012

    Figure Lengend Snippet: ATG7 Overexpression Inhibited p27 mRNA Transcription in Human BC Cells (A) UMUC3(shATG7#1), UMUC3(shATG7#2), and UMUC3(Nonsense) cells were cultured in 6-well plates until the cell density reached 70%–80%. Following synchronization for 12 hr, the medium was replaced with 10% FBS DMEM for another 12 hr. Then the cells were extracted for total RNA with TRIzol reagent. RT-PCR was used to determine p27 mRNA expression, whereas β-actin was used as an internal control. (B) Schematic representation of the p27 promoter region p27 KPNI and its deletion fragment SACII . (C) p27 promoter transcription activity was evaluated by using the two p27 promoter-driven luciferase reporters shown in (B). The results were normalized by internal TK activity, and the bars show mean ± SD from three independent experiments. The asterisk indicates a significant increase in comparison with vector control transfectant (*p

    Article Snippet: Total RNA was extracted using TRIzol reagent (Invitrogen) as described in the manufacturer’s instructions.

    Techniques: Over Expression, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing, Activity Assay, Luciferase, Plasmid Preparation, Transfection

    Pathways modulated in chronic HCV. RNA from liver biopsies from 8 HCV patients and normal donor livers were isolated using Trizol reagent and miRNA expression profile in the samples were analyzed using Illumina Bead array. The differentially expressed miRNA identified using Partek analysis were grouped based on the biological pathways that could be modulated.

    Journal: PLoS ONE

    Article Title: Hepatitis C Virus Induced miR200c Down Modulates FAP-1, a Negative Regulator of Src Signaling and Promotes Hepatic Fibrosis

    doi: 10.1371/journal.pone.0070744

    Figure Lengend Snippet: Pathways modulated in chronic HCV. RNA from liver biopsies from 8 HCV patients and normal donor livers were isolated using Trizol reagent and miRNA expression profile in the samples were analyzed using Illumina Bead array. The differentially expressed miRNA identified using Partek analysis were grouped based on the biological pathways that could be modulated.

    Article Snippet: Liver biopsy that were snap frozen obtained from HCV or control normal liver, were used to isolate RNA using Trizol reagent (Invitrogen, Carlsbad, CA) as per manufacturer's protocol.

    Techniques: Isolation, Expressing

    Increased miR200c decreases FAP-1 expression. Human liver fibroblasts were transfe cted with pre-miR200c miRNA or scrambled miRNA (200 nM) using Lipofectamine RNAiMax and stimulated with TGF-β (50 ng/mL). RNA was isolated using trizol reagent and expression levels of miR200c and FAP-1 were measured using pre-developed Taqman miRNA and mRNA assays respectively. The small RNA U6b (miRNA) and GAPDH (mRNA) was used as an endogenous control and relative levels was calculated by the ΔΔCt method. Bars represent the mean expression observed in 3 different experiments performed with 3 different fibroblasts. In order to further define the role of miR200c, we cotransfected fibroblasts with pre-miR200c and mir Vana® miRNA inhibitor for miR200c and analyzed for the expression levels of miR200c and FAP-1. Cotransfection with mir Vana® miRNA inhibitor (50 nM) resulted in restoration of the levels of miR200c and FAP-1 to the levels observed in the untreated fibroblasts. a) Relative expression levels of miR200c; b: Relative expression levels of FAP-1 at the mRNA level; c: Relative expression levels of FAP-1 at the protein level and d: Representative western blot analysis of FAP-1. Lanes 1) Fibroblasts; 2) Fibroblast + TGF-β; 3) Fibroblast + TGF-β+pre-miR-miR200c; 4) Fibroblast + TGF-β+ scrambled pre-miR; 5) Fibroblast + TGF-β+ pre-miR –miR200c+ mir Vana® miRNA inhibitor control; and 6) Fibroblast + TGF-β+ pre-miR –miR200c+ mir Vana® miRNA inhibitor miR200c.

    Journal: PLoS ONE

    Article Title: Hepatitis C Virus Induced miR200c Down Modulates FAP-1, a Negative Regulator of Src Signaling and Promotes Hepatic Fibrosis

    doi: 10.1371/journal.pone.0070744

    Figure Lengend Snippet: Increased miR200c decreases FAP-1 expression. Human liver fibroblasts were transfe cted with pre-miR200c miRNA or scrambled miRNA (200 nM) using Lipofectamine RNAiMax and stimulated with TGF-β (50 ng/mL). RNA was isolated using trizol reagent and expression levels of miR200c and FAP-1 were measured using pre-developed Taqman miRNA and mRNA assays respectively. The small RNA U6b (miRNA) and GAPDH (mRNA) was used as an endogenous control and relative levels was calculated by the ΔΔCt method. Bars represent the mean expression observed in 3 different experiments performed with 3 different fibroblasts. In order to further define the role of miR200c, we cotransfected fibroblasts with pre-miR200c and mir Vana® miRNA inhibitor for miR200c and analyzed for the expression levels of miR200c and FAP-1. Cotransfection with mir Vana® miRNA inhibitor (50 nM) resulted in restoration of the levels of miR200c and FAP-1 to the levels observed in the untreated fibroblasts. a) Relative expression levels of miR200c; b: Relative expression levels of FAP-1 at the mRNA level; c: Relative expression levels of FAP-1 at the protein level and d: Representative western blot analysis of FAP-1. Lanes 1) Fibroblasts; 2) Fibroblast + TGF-β; 3) Fibroblast + TGF-β+pre-miR-miR200c; 4) Fibroblast + TGF-β+ scrambled pre-miR; 5) Fibroblast + TGF-β+ pre-miR –miR200c+ mir Vana® miRNA inhibitor control; and 6) Fibroblast + TGF-β+ pre-miR –miR200c+ mir Vana® miRNA inhibitor miR200c.

    Article Snippet: Liver biopsy that were snap frozen obtained from HCV or control normal liver, were used to isolate RNA using Trizol reagent (Invitrogen, Carlsbad, CA) as per manufacturer's protocol.

    Techniques: Expressing, Isolation, Cotransfection, Western Blot

    Expression of Nox family homologs in adipose tissue and various cultured cells. (A) RT-PCR of Nox family homologs. Total RNA was prepared using the TRIzol reagent, and RT-PCR was performed with primers specific for human Nox1, Nox2, Nox3, Nox4, Nox5, Duox1, and Duox2 in human subcutaneous and omental adipose tissue, HepG2 hepatoma cells, and SV40-transformed human microvascular endothelial (HADMEC-5) cells using previously reported techniques . N1 to N5, Nox1 to Nox5; D1 and D2, Duox 1 and 2; G, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control; M, molecular size markers. (B) RT-PCR was performed using RNA isolated from differentiated 3T3-L1 adipocytes as described in Materials and Methods using primers for PCR analysis that were specific to murine Nox family homologs Nox1, Nox2, and Nox4. Control cell lines included CMT-93 (murine colon carcinoma cells), HL-60 (human leukemia cells), and MMC (murine mesangial cells). (C) Northern blot analysis of Nox4 mRNA expression in differentiated 3T3-L1 adipocytes. Total RNA was isolated from the indicated cells using the TRIzol reagent, and Northern blot analysis using Nox1-, Nox2-, and Nox4-specific cDNA probes or GAPDH as a loading and transfer control was performed as described in Materials and Methods.

    Journal:

    Article Title: The NAD(P)H Oxidase Homolog Nox4 Modulates Insulin-Stimulated Generation of H2O2 and Plays an Integral Role in Insulin Signal Transduction

    doi: 10.1128/MCB.24.5.1844-1854.2004

    Figure Lengend Snippet: Expression of Nox family homologs in adipose tissue and various cultured cells. (A) RT-PCR of Nox family homologs. Total RNA was prepared using the TRIzol reagent, and RT-PCR was performed with primers specific for human Nox1, Nox2, Nox3, Nox4, Nox5, Duox1, and Duox2 in human subcutaneous and omental adipose tissue, HepG2 hepatoma cells, and SV40-transformed human microvascular endothelial (HADMEC-5) cells using previously reported techniques . N1 to N5, Nox1 to Nox5; D1 and D2, Duox 1 and 2; G, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control; M, molecular size markers. (B) RT-PCR was performed using RNA isolated from differentiated 3T3-L1 adipocytes as described in Materials and Methods using primers for PCR analysis that were specific to murine Nox family homologs Nox1, Nox2, and Nox4. Control cell lines included CMT-93 (murine colon carcinoma cells), HL-60 (human leukemia cells), and MMC (murine mesangial cells). (C) Northern blot analysis of Nox4 mRNA expression in differentiated 3T3-L1 adipocytes. Total RNA was isolated from the indicated cells using the TRIzol reagent, and Northern blot analysis using Nox1-, Nox2-, and Nox4-specific cDNA probes or GAPDH as a loading and transfer control was performed as described in Materials and Methods.

    Article Snippet: For Northern analysis, total RNA was isolated from differentiated 3T3-L1 adipocytes using the TRIzol reagent (Invitrogen).

    Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Isolation, Polymerase Chain Reaction, Northern Blot

    Expression of Nox4 protein in 3T3-L1 adipocytes after transfection of siRNA oligonucleotides and expression of recombinant Nox4 mRNA and PTP1B protein in 3T3-L1 adipocytes following adenovirus-mediated gene delivery. (A) 3T3-L1 adipocytes were transfected with Nox4-specific (siRNA 1, 3, or both together) or a scrambled control siRNA oligonucleotide and reseeded in six-well plates . After 48 h of incubation, cell lysates were prepared and incubated with Nox4 polyclonal antibody to immunoprecipitate Nox4 protein. Immunoprecipitates were separated by SDS-PAGE and reblotted with Nox4 antibody as described in Materials and Methods. A representative blot demonstrating expression of Nox4 protein at ∼65 kDa and a reduction in Nox4 abundance following loading of specific siRNA oligonucleotides is shown. Protein expression for the insulin receptor β-subunit is shown as an experimental control. The bar graph shows mean data from replicate immunoblots quantitated using ImageStation 440 (Kodak). *, P = 0.05; **, P = 0.01 versus control cells transfected with the scrambled siRNA. (B) At 72 h following transduction of differentiated 3T3-L1 adipocytes with adenovirus encoding β-galactosidase or the indicated Nox4 constructs, total RNA was prepared using the TRIzol reagent and RT-PCR was performed with Nox4-specific primers as described in Materials and Methods. Nox4 was detected as a ∼550-bp band in the ethidium bromide-stained agarose gel. (C) Cell lysates were prepared from 3T3-L1 cells 72 h after transduction with control adenovirus encoding β-galactosidase, wild-type PTP1B, or a site-directed catalytically inactive (Cys215 S→er) PTP1B construct (mPTP1B). Proteins were separated by SDS-PAGE and immunoblotted with PTP1B antibody as described in Materials and Methods. The 50-kDa PTP1B protein band is indicated. Protein expression for the insulin receptor β-subunit is shown as an experimental control.

    Journal:

    Article Title: The NAD(P)H Oxidase Homolog Nox4 Modulates Insulin-Stimulated Generation of H2O2 and Plays an Integral Role in Insulin Signal Transduction

    doi: 10.1128/MCB.24.5.1844-1854.2004

    Figure Lengend Snippet: Expression of Nox4 protein in 3T3-L1 adipocytes after transfection of siRNA oligonucleotides and expression of recombinant Nox4 mRNA and PTP1B protein in 3T3-L1 adipocytes following adenovirus-mediated gene delivery. (A) 3T3-L1 adipocytes were transfected with Nox4-specific (siRNA 1, 3, or both together) or a scrambled control siRNA oligonucleotide and reseeded in six-well plates . After 48 h of incubation, cell lysates were prepared and incubated with Nox4 polyclonal antibody to immunoprecipitate Nox4 protein. Immunoprecipitates were separated by SDS-PAGE and reblotted with Nox4 antibody as described in Materials and Methods. A representative blot demonstrating expression of Nox4 protein at ∼65 kDa and a reduction in Nox4 abundance following loading of specific siRNA oligonucleotides is shown. Protein expression for the insulin receptor β-subunit is shown as an experimental control. The bar graph shows mean data from replicate immunoblots quantitated using ImageStation 440 (Kodak). *, P = 0.05; **, P = 0.01 versus control cells transfected with the scrambled siRNA. (B) At 72 h following transduction of differentiated 3T3-L1 adipocytes with adenovirus encoding β-galactosidase or the indicated Nox4 constructs, total RNA was prepared using the TRIzol reagent and RT-PCR was performed with Nox4-specific primers as described in Materials and Methods. Nox4 was detected as a ∼550-bp band in the ethidium bromide-stained agarose gel. (C) Cell lysates were prepared from 3T3-L1 cells 72 h after transduction with control adenovirus encoding β-galactosidase, wild-type PTP1B, or a site-directed catalytically inactive (Cys215 S→er) PTP1B construct (mPTP1B). Proteins were separated by SDS-PAGE and immunoblotted with PTP1B antibody as described in Materials and Methods. The 50-kDa PTP1B protein band is indicated. Protein expression for the insulin receptor β-subunit is shown as an experimental control.

    Article Snippet: For Northern analysis, total RNA was isolated from differentiated 3T3-L1 adipocytes using the TRIzol reagent (Invitrogen).

    Techniques: Expressing, Transfection, Recombinant, Incubation, SDS Page, Western Blot, Transduction, Construct, Reverse Transcription Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis

    CO-EtOAc regulates the expression of lipid metabolism-related genes in FL83B hepatocytes. Total RNAs were extracted using Trizol reagent and mRNA was measured using qRT-PCR. Relative mRNA levels were normalized to the housekeeping gene, r18S. The fold changes relative to the control were calculated using the ΔΔCT method for mRNA expression levels of fatty acid transporter genes, CD36 and FABP ; lipogenic genes: SREBP-1c , FAS , and ACC1 ; fatty acid oxidation genes: PPAR-α, CPT-1 , and ACOX . All experimental groups had been treated with the same concentration of DMSO. ( A ) Cells were incubated with different concentrations (50–400 μg/mL) of CO-EtOAc. ( B ) Cells were incubated with 600 μM OA in the presence or absence of different concentrations (50–400 μg/mL) of CO-EtOAc. All results are presented as the mean ± SEM of three independent experiments. Values are expressed as relative fold change compared with the control group, which is set as 1. * p

    Journal: Scientific Reports

    Article Title: Chinese olive extract ameliorates hepatic lipid accumulation in vitro and in vivo by regulating lipid metabolism

    doi: 10.1038/s41598-018-19553-1

    Figure Lengend Snippet: CO-EtOAc regulates the expression of lipid metabolism-related genes in FL83B hepatocytes. Total RNAs were extracted using Trizol reagent and mRNA was measured using qRT-PCR. Relative mRNA levels were normalized to the housekeeping gene, r18S. The fold changes relative to the control were calculated using the ΔΔCT method for mRNA expression levels of fatty acid transporter genes, CD36 and FABP ; lipogenic genes: SREBP-1c , FAS , and ACC1 ; fatty acid oxidation genes: PPAR-α, CPT-1 , and ACOX . All experimental groups had been treated with the same concentration of DMSO. ( A ) Cells were incubated with different concentrations (50–400 μg/mL) of CO-EtOAc. ( B ) Cells were incubated with 600 μM OA in the presence or absence of different concentrations (50–400 μg/mL) of CO-EtOAc. All results are presented as the mean ± SEM of three independent experiments. Values are expressed as relative fold change compared with the control group, which is set as 1. * p

    Article Snippet: RNA extraction and qRT-PCR Total RNA was isolated using Trizol Reagent (Roche; Basel, Switzerland) according to manufacturer’s protocol and quantified by spectrophotometry at 260 nm.

    Techniques: Expressing, Quantitative RT-PCR, Cycling Probe Technology, Concentration Assay, Incubation

    CO-EtOAc attenuates hepatic lipid accumulation in C57BL/6 mice treated with 60% high-fat diet (HFD). C57BL/6 mice were fed a high fat diet (HFD) for 21 weeks and treated with CO-EtOAc in the last 4 weeks. The control group was administered a low-fat diet (LFD). At the end of the experiment, all mice were killed by CO 2 anaesthesia. The liver samples were fixed in 10% formalin for 24 h, embedded in paraffin, sectioned, and stained with haematoxylin and eosin (H E). ( A ) Representative photographs of the liver appearance (upper) and the abdominal fat (lower) of mice among different groups are shown. ( B ) Histological features of the lipid accumulation in the livers of C57BL/6 mice. Representative photographs are shown with 100X magnification. All scale bars indicate 50 μm. ( C ) Total RNAs were extracted using Trizol reagent and mRNA was measured using qRT-PCR. Relative mRNA levels were normalized to the housekeeping gene, r18S. The fold changes relative to the control were calculated using ΔΔCT method for mRNA expression levels of fatty acid transporter genes, CD36 and FABP ; lipogenic genes: SREBP-1c , FAS , and ACC1 ; fatty acid oxidation genes: PPARα, CPT-1 , and ACOX . The levels of protein expression of ( D ) phosphorylated and total AMPKα, phosphorylated and total ACC1, and FAS ( E ) SREBP-1c ( F ) CPT-1 ( G ) PPAR-α were measured by western blot analysis. The intensity of the protein band was normalized against the internal control β-actin. Histograms depict the quantitative analysis of the results and the value for the control group was set as 1. All results are expressed as the mean ± SEM of three independent experiments. * p

    Journal: Scientific Reports

    Article Title: Chinese olive extract ameliorates hepatic lipid accumulation in vitro and in vivo by regulating lipid metabolism

    doi: 10.1038/s41598-018-19553-1

    Figure Lengend Snippet: CO-EtOAc attenuates hepatic lipid accumulation in C57BL/6 mice treated with 60% high-fat diet (HFD). C57BL/6 mice were fed a high fat diet (HFD) for 21 weeks and treated with CO-EtOAc in the last 4 weeks. The control group was administered a low-fat diet (LFD). At the end of the experiment, all mice were killed by CO 2 anaesthesia. The liver samples were fixed in 10% formalin for 24 h, embedded in paraffin, sectioned, and stained with haematoxylin and eosin (H E). ( A ) Representative photographs of the liver appearance (upper) and the abdominal fat (lower) of mice among different groups are shown. ( B ) Histological features of the lipid accumulation in the livers of C57BL/6 mice. Representative photographs are shown with 100X magnification. All scale bars indicate 50 μm. ( C ) Total RNAs were extracted using Trizol reagent and mRNA was measured using qRT-PCR. Relative mRNA levels were normalized to the housekeeping gene, r18S. The fold changes relative to the control were calculated using ΔΔCT method for mRNA expression levels of fatty acid transporter genes, CD36 and FABP ; lipogenic genes: SREBP-1c , FAS , and ACC1 ; fatty acid oxidation genes: PPARα, CPT-1 , and ACOX . The levels of protein expression of ( D ) phosphorylated and total AMPKα, phosphorylated and total ACC1, and FAS ( E ) SREBP-1c ( F ) CPT-1 ( G ) PPAR-α were measured by western blot analysis. The intensity of the protein band was normalized against the internal control β-actin. Histograms depict the quantitative analysis of the results and the value for the control group was set as 1. All results are expressed as the mean ± SEM of three independent experiments. * p

    Article Snippet: RNA extraction and qRT-PCR Total RNA was isolated using Trizol Reagent (Roche; Basel, Switzerland) according to manufacturer’s protocol and quantified by spectrophotometry at 260 nm.

    Techniques: Mouse Assay, Staining, Quantitative RT-PCR, Expressing, Cycling Probe Technology, Western Blot